Population Dynamics Analysis of Chromochloris zofingiensis: A Flow-Cytometry-Based Approach

The design and optimization of microalgae processes are usually focused on maximizing biomass productivity, neglecting the impact of cell-to-cell heterogeneity. Flow cytometry (FCM) represents a powerful and high-throughput tool for analyzing and examining microalgae intrinsic characteristics, such as their physiology, metabolism and response at the single-cell level. The aim of this work is to develop a novel FCM sensor-based single-cell analysis method to monitor and study the effect of several process conditions, mainly variations of light spectral composition (blue, red and green), nitrogen depletion and moderate osmotic stress conditions (0.2 M NaCl), on the subpopulation structure and dynamics of the green microalgae Chromochloris zofingiensis, a natural source for lipids, proteins and carotenoids. The FCM procedures developed in this study proved to be effective for monitoring the population dynamics of microalgae, demonstrating how the process conditions have a direct and significant impact on population heterogeneity of C. zofingiensis on a single-cell level. Cell division was found to be adversely affected by the moderate osmotic stress (N⁺S⁺), nitrogen depletion (N^-), and their combined occurrence (N^-S⁺), independent of the light spectral composition used for culture illumination. In terms of cell-to-cell heterogeneity, a higher proportion of large cells (~20 µm) was observed under green light across all conditions with 21%, 29%, 35% and 52% under N^-, N^-S⁺, N⁺S⁺ and N⁺ conditions, respectively, followed by red light combined with osmotic stress (46%), whereas blue light consistently led to a predominance of smaller cells (≤4 µm) with 30%, 47%, 50% and 55% under N⁺S⁺, N⁺, N^-S⁺ and N^- conditions, respectively.